TFIHOHBOSIS Pergamon
RESEAHCH 16; 1?5-189 Press Ltd.1979. Printed
in Great
Britain
A COACLZO-lUDIOIMMUNOMBTRIC ASSAY FOR THE QUANTITXTION OF FIBRIN MONOMER IN HDMAN PLASMA. PRINCIPLES AND DEVELOPZNT OF THE METHOD
3.
Felez*, E.F. Plow, B. Wiman and D. Collen
Center for Thrombosis and Vascular Research Department of Medical Research University of Leuven, Belgium
(Received
26.12.1978; in revised form 23.5.1979. by Editor M.J. Larrieu)
Accepted
ABSTRACT When a mixture of '251-labeled human fibrin monomer (HFM) and human plasma or purified fibrinogen (HFg) is added to insolubilized bovine fragment D (BFD-Sepharose), the amount of HFM which binds to BFD-Sepharose is proportional to the amount of HFM and BFD-Sepharose and inversely proportional to the amount of fibrinogen in the test tube. With 15-fold molar excess of insolubilized BFD over HFg and 2 to IO percent of HFM (compared to HFg) in the test system about 25 percent of the HFM binds to insolubilized BFD. The distribution ratio of labeled HFM is however almost independent of the concentration of HFM, and can therefore not be used to quantitate HFM in plasma. When purified 1311-labeled antibodies to HFg are added to BFDSepharose to which 1251-labeled HM is bound, the binding of 1311 is proportional to the amount of HFM on the gel. This indicates that labeled antibodies can be used to quantitate HFM bound to BFD-Sepharose. Under the experimental conditions studied (20 ~1 plasma containing 50 ug HFg and 0 to 5 ug HFM incubated with 0.25 mg insolubilized BFD), 0.6 - 0.8 percent of the plasma fibrin(ogen) and 25 percent of the HFM bind to the gel. Thus addition ?5,3_percent HFM to plasma results in a doubling of the amount of I labeled anti-HFg Ig bound to BFD-Sepharose. This test tube method for the quantitation of HFM in plasma has an analysis time of about 5 hours in its present version and a large number of samples can be handled at the same time. It may therefore constitute a useful alternative to the affinity chromatographic method for measuring HFM in plasma.
*
On leave from Servicio Hematologia, Hospital S. Pablo, Barcelona, Spain. Abbreviations used : HFg : human fibrinogen; BFg : bovine fibrinogen; HFM : human fibrin monomer; BFD : bovine fraqnen: D
CRIXA
FOR FIBRIN
VOf.l6,NO.L/’
INTRODUCTION The presence of fibrin monomer in plasma provides primary and direct evidence for the activation of the coagulation system leading to fibrin formation. Detection of fibrin, either as circulating monomer or in complexes with fibrinogen , is diagnostic of disseminated intravascular coagulation and an indicator for the “hypercoagulable state” (1). The specific detection and quantitation of fibrin monomers by affinity chromatography on insolubilized fibrinogen (2-4) is based on the original description of Heene and Matthias (5). In methodological studies (6,7) we have demonstrated that insolubilized fragment D may be substituted for insolubilized fibrinogen and constitute a more specific adsorbant for fibrin monomer from human plasma. The affinity chromatographic procedures, performed on columns, have some practical disadvantages which hamper their routine application. Large matrix to sample ratios are used to obtain near-quantitative binding of fibrin and extensive washing is required to remove co-adsorbed fibrinogen from the columns before dissociating the soluble fibrin for further analysis. Thus in general the analysis time is about one day and only a few samples can be handled at the same time. In view of these disadvantages, we have tried to elaborate a test tube method for the quantitation of soluble fibrin in human plasma. The present studies demonstrate that under appropriate conditions the distribution of fibrin monomer between soluble fibrinogen and insolubilized bovine fragment D is relatively independent of fibrin monomer concentration. This permits the use of labeled specific antibodies to human fibrinogen to precisely quantitate fibrin monomers bound to insolubilized fragment D in a highly sensitive assay system. MATERIALSAND METHODS Fibrinogen Human fibrinogen (grade L) (HFg) and bovine fibrinogen (BFg) were gifts from Kabi AB, Stockholm, Sweden (courtesy of Dr. B. Strindberg). Purification
of BFD
Two g of BFg were dissolved in 100 ml 0.05 M phosphate - 0.10 M NaCl and digested with plasmin (0.5% buffer, pH 7.5, containing 0.04% Na axide, W/W) obtained by activation of purified human plasminogen with insolubilized urokinase (8). The digestion was carried out at 4’C overnight and terminated by addition of aprotinin (Trasylol, Bayer, Leverkusen, W. Germany) (2,000 kallikrein inhibitor units (KIU) per mg plasmin). using a combination of affinity chroBFD was isolated from the digest, matography on insolubilized thrombin-treated BFg (bovine fibrin monomerSepharose) (9) followed by gel filtration on Sephadex G-150 in 0.1 M NaHC08 Sodium dodecylsulphate polyacrylamide gel electrophore- 2 M NaCl, pH 8.0. sis of the BFD preparations used in this study revealed essentially one band with a molecular weight of approximately 100,000, very similar to human fragment D previously described (9).
CRIYA
vo1.16,xo.1/2
Preparation
of
insolubilized
FOR
BFD for
FIBRIN
affinity
177
chronacography
Purified BFD was coupled to Sepharose 4 B (Pharmacia Fine Chemicals, Uppsala, Sweden) by the CNBr procedure essentially as described by Heene and Matthias (5). The substitution ratio was on average 12-14 mg BFD per ml gel (settled volume). The gel was washed with 0.05 M Tris.H3P04 - 0. I M NaC1 0.01 M EDTA - 0.001 M TAMe, buffer pH 7.4, containing 0.04% Na azide, 1X bovine serum albumin and IO KIti aprotinin/ml (buffer A), and suspended in approximately two volumes of the same buffer to obtain a BFD concentration of 5 mg per ml suspension. Purification
of
specific
antibodies
to
HFg
Rabbits were immunized by multiple site injection of 0.1 mg of purified HFg suspended in Freund adjuvant (complete adjuvant on the first injection, incomplete adjuvant on subsequent injections) at weekly intervals for three weeks. Serum obtained from late Booster injections were given bimonthly. bleedings of individual rabbits was used for isolation of the antibodies. Purified antibodies ?o HF8 which do not cross-react with BFD were isolated by absorption of the antiserum with insolubilized BFg followed by immunoabsorption on insolubilized HFg and elution of the specific antibodies, The titers of the which were finally again absorbed with insolubilized BFD. antisera were determined by hemagglutination as described by Merskey et al. using tanned red cells coated with purified HFg (I mg protein incuba(IO), ted per 100 ml cell suspension) or purified BFD (2 ag fragment D incubated with 100 ml cell suspension) . In a typical exp*riment, 50 ml of rabbit antiserum to HFg with a titer of l/8000 as assayed with red cells coated with HFg and a titer of l/500 asthrough a column (I .5 x sayed with the red cells coated with BFD, was passed 25 cd of BFg-Sepharose (substitution ratio 17.4 mg fibrinogen/ml gel), equilibrated with 0.05 M phosphate - 0.1 ?! NaCl buffer pH 7.5, containing 0.04X sodiumazide and IO KIU aprotinin/ml at room temperature and a flow rate of 40 ml per hour. The adsorbed antibodies were eluted with 0.1 M glycine. HCl pH 2.8, the column reequilibrated and the solid phase absorption repeated twice on the filtrate. after concentration to its The resulting antiserum, original volume had a titer of l/1000 assayed with red cells coated with HFr and c I/? with red cells coated with BFD. The specific antibodies to HFg were isolated from this absorbed antiserum by immunoabsorption on a column (0.9 x 12 cm) of HFg-Sepharose (substitution ratio 19 mg/ml of settled gel), using 0.1 PI glycine.HCl, pH 2.8, for dissociation of the antigen-antibody complexes. The eluted protein was dialyzed against 0.05 M phosphate - 0.1 M NaCl buffer pH 7.5, containing 0.042 sodiumazide and IO KIU aprotinin/ml, and after concentration by vacuum dialysis, applied to an Ultrogel AcA34 column (2.5 x 40 cm). The fractions containing agglutinating activity towards red cells coated with HFg were pooled and concentrated to 20 ml. This antibody solution had an absorbancy of 0.19 at 280 run, a titer of l/1000 in the assay with red cells coated i;ith HFg and a titer of < I 2 In the assay with BFD cells. The purified antibodies were labe 1 ed wi th I as described below with specific activities which attained uo to 1 mCi/m6’brotein. After iodination another absorption was performed by passing the antibody solution through a 0.9 x 3 cm column of BFD-Sepharose (Substitution ratio 13 mg per ml gel). This procedure did not affect the protein concentration or titer of the antibody solution with HFg coated red cells but reduced the binding of labeled antibodies to insolubilized BFD to less < 1 .5 percent.
Preparation
of
:iF?I
HFM was prepared by dissolving HFg in 0.25 l! phosphat2 - :?. i’? Y ::a:1 containing IO KIU aprotinin/ml, to a final con.-entra0.005 M EDTA, pH 7.5, ,_ tion of 1 to 7 mg per ml. Thrombin (Topostasine, Roche, Basel, Switzerland) per ml solution at room was added to a concentration of I to 2 NIH units temperature and the fibrin collected by winding it onto a glass rod. Xfter 30 min the clot was removed, dried on filter paper, washed twice with buffer and dissolved in 0.05 X sodium acetate - 2 X ?;aBr, buffer pH 5.3, containing 10 KIU aprotinin per nl. The dissolved fibrin xas reclotted by diluting the NaBr solution ten-fold with 0.05 ?! phosphate - 0. i !I XaCl - 0.005 ?f EDT_‘ buffer pH 7.5 and the clot was again redissolved in 0.05 X sodium acetate - 3 X SaBr buffer, pH 5.3. The fibrin concentration in the final solution was determined from its absorbancy at 280 nm (Al% = 15) and purified antithrombin III (obtained 28Q n$ from Kabi AB, Stockholm, we en) and heparin (Liquemine, Roche, Basel, Switzerland) were added in ten-fold molar excess to thrombin in order to insure inhibition of residual thrombin. 1251-labeled HFX was obtained The of labeled and unlabeled HFg. solutions averaged 20 uCi per mg.
in the specific
same way, activity
starting of the
from mixtures labeled HP>:
Radioiodination Proteins were radioiodinated by chloramine T oxidation using a reduced Initially 0.5-I .O mCi carrier free Sal251 or Xa13’I volume procedure (11). (IRE, Fleurus, Belgium) were added to 100 ug protein at 1.0 mg/ml in 0.5 11 The reaction was initiated by addition of 20 ;g sodiumphosphate pH 7.3. chloramine T and proceeded for 3 min at room temperature at which time 22 ug sodium disulfite was added followed by RI and bovine serum albumin to final ‘*‘I or 1311 were removed by exconcentrations of 0.1 mg per ml. Unbound Specific activities of 1 mCi/mg were routinally achieved tensive dialysis. and precipitability of radioactivity in 10% trichloroacetic acid generally exceeded 97%. Quantitation solubilized
of the BFD
distribution
of
labeled
HFX between
soluble
HFg and
in-
the distribution of HFM between soluble HFg and Unless stated otherwise, insolubilized BFD was measured by adding successively to 6 ml polystyrene tubes : 1) 10 to 200 ul titrated human plasma or purified HFg solution 2) 20 ~1 of a mixture of labeled and (fibrinogen concentration 2.5 mg/ml), unlabeled HFX dissolved in 0.05 M acetate - 2 Y NaBr pH 5.3 (amount of fibrin incubation for I hour at room temperature sufadded 0.5 to IO ug) , 3) after ficient diluent consisting of 0.05 M Tris.H PO4 - 0.10 M NaCl - 0.01 ?! EDTA IX bovine serun albumin - 0.001 M TAMe, pH 7.4, containing 0.04% so a-iumazide, and IO KIU aprotinin per ml (buffer A) was added to obtain a final volume of 0.5 ml, and 4) after 10 min incubation 50 to 200 ul suspension of BFD-Sephacontaining 5 mg of BFD per ml suspension. rose, The mixture was stirred for I hr at room temperature and then centriThe gel was washed 5 times by suspending it in fuged for 2.5 min at 3000 g. 1 ml buffer A and stirring for approximately 5 min at room temperature. The HFM which remained ass?Siated with the insolubilized BFD was then calculated During the 4th and the 5th wash, IO to 20 percent from measurements of I. of the radioactivity which remained associated with the gel was removed.
CRI%
Quantitation of EM, to HFg.
FOR
FIBRIN
bound to insolubilized 8FD ;lith '3'I-labeled antibodies
The HFH containing BFD-Sepharose was resuspended in 0.: ml buffer A, 100 21 of a mixture of !3iI-labeled and unlabeled antibodies to HFg was added and the mixture stirred for I hr at room temperature. The labeled antibodies were diluted with unlabeled antibodies in such a vay that about I5 percent of the label (1500 cpm) was bound by 0.25 mg insolubilized BFD containing 1 ug HFX. The mixture was stirred for I hour at room ;;yperature and I. Control the gel ijaswashed 5 times with I ml buffer to remove unbound experiments without RFg, HFX or both added to the BFD-Sepharose were run simultaneously. With the fifth wash only negligible amounts of 13'1 were eliminated. Labeled antibody displacement curves The optimal dilution of labeled with unlabeled antibodies for the quantitation of HFM bound to insolubilized BFD was determined in the following way. Standard amounts of HFN bound to BFD-Sepharose (0.25 mg BFD) were incubated with 1311-labeled antibodies to HFg which were progressively diluted in cold antibodies. The HFN substituted BFD-Sepharose was obtained by incubating l251-labeled HFM in the absence of HFg with BFD-Sepharose and the amount of HFM bound to BFD calculated from 1251 measurements. Binding ratios of 1.0, 0.5 and 0.25 pg of HFX per 0.25 mg of insolubilized BFD were analyzed. The gel was suspended in sufficien t bclffer A to obtain a final volume .>f0.50 ml, the mixture of labeled (representing 10,000 cpm) and unlabeled antibodies was added and the suspension stirred for one hr at room temperature. The gel was then isolated by centrifugation, washed 5 times with I ml volumes of buffer A and the '311 associated with the gel was then measured. Recycling of BFD-Sepharose 125 I-labeled HFM and BFD-Sepharose which had been in contact with both i311-labeled anti-HFg Ig was collected and stored in buffer A. When sufficient gel had accumulated, it was poured in a column and washed with 0.05 M sodium acetate - 2 m NaBr buffer pH 5.3 for at least 24 hr. This resulted in removal of 80-90 percent of both 1251 and I311 associated with gel without loss of binding capacity for 125I-HFX. RESULTS Distribution of human fibrin monomer between soluble HFg and insolubilized BFD When 20 ~1 mixtures of labeled and unlabeled (0.5 to 2.5 ug) HFM were added to 200 ~1 BFD-Sepharose (1 mg BFD) suspension in a total volume of 0.5 ml and the suspension stirred for I hr at room temperature about 90 per-cent of the labeled material remained associated with the gel after washing (Fig. IA). When 200 pl of purified HFg solution or human plasma were preincubated for I hr at room temperature with 20 ul mixtures of labeled and unlabeled HFM (1 to 10 ug) and then the BFD-Sepharose was added, the percentage of labeled material which bound to BFD decreased with increasing amounts of HFM and moreso with increasing concentration of HFg (Fig. IA). The total amount of HFM which remained associated with the insolubilized BFD, calculated from the experiments in Fig. IX is represented in Fig. iB. The results demonstrate that the amount of HM associated with the BFD was
CRI;ttl.
189
proportional concentration. solubilized
FOR
TiOl.lS,SO.l/2
FIBRI?;
to
the HE?! concentration and inversely The latter findings indicate that BFD compete with each other for !!FX.
?ro?or:i~al the soluble
Wg
to the :iCg and the in-
60 I
OL
G
1
3 HFM
3
lo ADDED
HFM
(~91
FIG.
la ADDED
(w)
I
Binding of fibrin monomer to insolubilized bovine fragment D. Experimental conditions : 200 ul fibrinogen solution of different concentra tion was incubated with 20 ul of a mixture of labeled and unlabeled fibrin 1 hr at room temperature. Then 200 ul suspension of insolubimonomer for lized fragment D (representing 1 mg fragment D) was added and after incubation for I hr the unbound label was removed by washing the gel 5 times with 1 ml buffer. The amount of radioactivity associated with the gel was measured and expressed as percentage of the total (A) and converted into ug human fibrin monomer bound (B) . The data represent means + SD from four separate experiments. The amount of HFM associated with the gel exhibited a linear dependence on the total amount of insolubilized BFD present (Fig. 2). This relationship was only marginally affected by fibrinogen concentration. The influence of the incubation time on the binding of fibrin monomer is shown in Fig. 3. Maximum binding was obtained after about 2 hours and remained constant between 2 and 4 hours.
CRI?Q~ FOR
I
0.25 INSOLUBILIZED
1Sl
FIBRIX
I
I
0.50 8FD
1 (mg)
FIG. 2 Influence of the amount of insolubilized bovine fragment D on the binding of human fibrin monomer. Experimental conditions : IO0 ~1 fibrinogen solution was incubated with IO ul mixtures of labeled and unlabeled fibrin monomer for 1 hr at room temperature. Then buffer and insolubilited bovine fragment D suspension were added to a final volume of 0.5 ml. The total amount of gel in each tube was adjusted with non-substituted gel to a volume of 200 ~1. (*I : 3 ug fibrin monomer and 500 pg fibrinogen in the assay system (a) : 3 up fibrin monomer and 250 ug fibrinogen (0) : IO ug fibrin monomer and 500 u'gfibrinogen in the assay system @I) : 10 g fibrin monomer and 250 ug fibrinogen
These results establish the characteristics of the affinity system when an initial distribution between '251-HFM and HFM and fibrinogen is permitted prior to addition of the BFD-Sepharose. Under these conditions, binding of HFM to the beads is dependent upon : a) HFM, b) fibrinogen, c) BFD beads, and d) time. In plasma samples for analysis, this condition would, however, not exist since an initial interaction of HFM in the plasma with plasma fibrinogen would occur prior to the addition of the '251-HFM. Therefore, the characteristics of the affinity system was examined whe HFX and fibrinogen were preincubated for one hour prior to the addition of iY5I-HFM. The results shown in Figure 4 demonstrate that such preincubation markedly affects the data observed. With such preincubation, the binding of labeled HFM to the EFD beads was no longer dependent upon HFM or fibrinogen concentration. This is in marked contrast to the results observed in Figure IA where, without preincubation, binding was dependent upon both these variables. Presumably, the dissociation constant of H Py5ard fibrinogen is low, and an effective reequilibration between HFM and I HFM is not achieved within one hour. Prolonged incubation with '251-HFM or high concentrations of the label should yield results similar to Figure IA but that would preclude a rapid or sensitive analysis. Therefore, it does not appear possible to quantitate HF?! in plasma by the addition of trace amounts of labeled HFY.
CRIXk
182
FOR
FIBRIX
VOl.lb,XO.l/2
/
g
;/* I / olY~ 1
??
INCUBATION FIG.
4
2 TIME
IN
HOURS
3
Influence of incubation time on the binding of labeled fibrin monomer to insolubilized bovine fragment D. Experimental conditions : 100 ~1 fibrinogen solution was premixed with IO ~1 labeled fibrin monomer for I hr at room bovine fragment D and buffer up to a temperature. Then I mg insolubilized final volume of 0.5 ml was added. After different incubation times the gel was washed 5 times and the amount of fibrin monomer associated with the gel quantitated by isotope measurements. monomer and 1500 pg fibrinogen in the assay (*I : IO ng fibrin 150 ug fibrinogen in the assay I ug fibrin monomer and (0) :
Quantitation of HFM, adsorbed specific antibodies to HFg
to
insolubilized
Addition of labeled HFM cannot Therefore a radioimmunometric assay adsorbed HFM.
BFD with
be used to measure was elaborated for
the
use
of
labeled
HFM bound to BFD. the quantitation of
Antibodies raised against HFg were immunochemically purified on insolunon-reactive with insolubilized BFD as described bilized HFg, and rendered When these labeled antibodies were added to under naterials and methods. 0.25 mg insolubilized HFg, between 30 and 60 percent was bound, whereas under comparable experimental conditions only I to 1.5 percent bound to 0.25 mg insolubilized BFD and 0.4 to 0.7 percent associated with an equivalent The antibodies therefore seem to be specific amount of unsubstituted gel. enough for quantitation of the HFM-bound to insolubilized BFD.
.
For quantitative analysis, labeled antibody displacement curves were constructed using HFM bound to insolubilized BFD as ligand and 1311-labeled anti-HFg Ig diluted with unlabeled antibodies as described under materials The results obtained in a typical experiment are shown in and methods Whereas 60 percent of the labeled antibodies bound to 250 ug HFM, Fig. 5. about 22 percent was associated with the BFD gel containing I u!p,9FM and 11
CRIMA
vo1.16,80.1/‘2
FOR
133
FIBRIS
1000 pg Fg f 270 rrg Fg f
, 1
I 3
10 HFM
ADDED
FIG.
(pg)
4
Binding of fibrin monomer to insolubilized bovine fragment D. Sxpcrlmental conditions : 200 ul fibrinogen solution of different concentration was incubated with IO ul of unlabeled fibrin monomer for 1 hr followed by addition of a trace amount of labeled fibrin monomer and further incubation I hr. for Then 200 ~1 suspension of insolubilited fragment D (representing 1 mg fragment D) was added and after incubation for I hr the unbound label was The amount of radioacremoved by washing the gel 5 times with I ml buffer. tivity associated with the gel was measured and expressed as percentage of the total. The data represent means -+ SD from four separate experiments.
percent with the gel containing 0.25 ug HF!+. These values are corrected for the binding of I .5 percent 1311 to 0.25 mg insolubilized BFD. Non-immune Ig did not cause displacement of labeled antibodies indicating that their binding is due to reaction with adsorbed HFM. For subsequent quantitation of HFX in plasma or purified fibrinogen solutions a labeled antibody dilution of 3.5 times was used. Calibration curves for the quantitation of HM bound to BFD-Sepharose were constructed by incubation of increasing amounts of a mixture of ‘251labeled and unlabeled liF!I (0.25 to 7 -g.) with 0.25 mg insolubilized BFD.
In
CRIWL
b
I
1 1.5 DILUTION
FOR
FIBRIS
t
3.5 OF 13’1 LABELED
13.5 ANTI- HFG
lg
FIG. 5 Antibody displacement curves of 1311-labeled anti-HFg Ig from HFM bound to BED-Sepharose. Experimental conditions : 0.25 mg insolubilized BFD to which 0.25 to 1 ng ~~~n:~In~~~n~ow~~oi~~~bt5~d with dilutions of.10 ~1 1311-labeled anti-HFg Ig I) in unlabeled antibodies for I hr at room temperature in a f&al volume of 0.5 ml. The unbound label was removed by 5 washings with 1 ml buffer and the '3*1 quantitated.
the absence of fibrinogen in the solution 70 to 85 percent of this material bound to the BFD-Sepharose. The washed gels were then incubated with labeled antibodies and the percent 13'1 bound quantitated as described above. An almost linear relationship was found between the log of HFM bound to BFDSepharose and the percentage labeled antibodies bound (Fig. 6). HFM was quantitated in human plasma in the following way. Fresh human plasma from two healthy subjects with a fibrinogen concentration of 2.5 mg per ml (20 ul) was mixed with 10 ul 0.05 M sodium acetate - 2 M NaBr pH 5.3 containing a mixture of labeled and unlabeled HFM (0 to 5 ug HFM) and 0.42 ml buffer A was added followed after approximately 10 min incubation by 50 ~1 BFD-Sepharose suspension (0.25 mg BFD). The mixture was stirred for I hr at room temperature and the gel washed 5 times for 5 min at room temperature with I ml buffer A. Incubation in the presence of 0.4 M urea or 0.i.M NaBr, or washing with buffer A with pH adjusted to 6.0 or 8.0 ot with buffer A containing I M NaCl did not alter the results significantly.
CRI?l.A
Vol.lf,So.1/2
FOR
FIBRIS
1Sj
T
I-
c-
I
I
.
0.1
02 HFM
a5 BOUND
1 TO
2
5
BFD-SEPHAROSE
10 tyg)
131 Quantitation of “FM bound to BFD-Sepharose with I-labeled anti-HFg Ig. Experimental conditions : see text. o and * represent curves obtained with antibodies purified from two different antisera; the data represent means + SD from triplicate runs.
The washed gels were suspended in 0.5 ml buffer A and a mixture of ‘3’1_ labeled (10,000 cpm) and unlabeled (dilution factor 3.5) anti-HFg Ig addfg’ The suspension was stirred for I hr at room temperature and the unbound I removed by washing the gel 5 times with I ml buffer for 5 min at room temperature. The bound isotope was then measured and the corresponding amount of HFM bound to BFD-Sepharose calculated from the calibration curve in Fig. 6. The results obtained with the plasma from two healthy syjjects are summarized in Fig. 7. In the absence of added HFM the amount of I bound to the gel corresponded to 0.3 and 0.4 ug, representing 0.6 to 0.8 percent of the fibrinogen. In the presence of added ‘251-labeled HFM the W3 bound to the gel increased proportionally to the amount of I-HFM associated with the gel and corresponded to the amount calculated from the calibration
i-01.
p
;6 ,x0.1/2
5d
!/ 8 z s Y G
0
0.5
ID
12’1 HFM-BOUND
15
TO BFD-SEPHAROSE
(m)
FIG.? Correlation between the amount of soluble fibrin in human plasma determined from measurements of added 1251-labeled HFM and from the binding of 13'1labeled anti-HFg Ig. The data are obtained with plasma from two different healthy subjects.
curve in Fig. 6 (the lines in Fig. 7 are drawn at an angle of 45"). These findings suggest that the quantitation of soluble fibrin in human plasma by way of I-labeled antibodies to HFg following adsorption of the plasma with insolubilized BFD may be a valid approach. DISCUSSION Affinity chromatography on insolubilized fibrinogen has been used for the quantitation of soluble fibrin in human plasma. This analysis may be of value for the diagnosis of in vivo coagulation but its clinical application is limited by the fact that column chromatography is time consuming and only one sample can be analyzed per column at one time. In order to circumvent these disadvantages we have tried to elaborate a test tube version based on the affinity between fibrin monomer and high molecular weight fragment D (6). As one might expect, the soluble fibrinogen in plasma competed with the insolubilized fragment D for the fibrin monomer resulting in incomplete transfer of fibrin monomer from fibrinogen to the insolubilized fragment D. Such competition between insolubilized fibrin monomer and soluble fibrinogen for fibrin monomer present in plasma has previously been described by Largo et al. (12). At a 15-fold molar excess of fragment D over fibrinogen which contained
Vol.f6,?io.1/2
CRI?U. FOR
FIBRIN
18;
1251-labeled fibrin monomer, approximately 25 percent of the 10 percent fibrin monomer was transfered to the insolubilized ligand whereas less than 1 percent of the fibrinogen remained associated with the gel after washing. No differences were observed between insolubilized human fragment D (data not reported) or bovine fragment D (BFD). The transfer of human fibrin monomer (HEM) to BFD thus is sufficiently high and the binding of human fibrinogen (HFg) sufficiently low to allow quantitation of HFX in plasma from the amount bound to insolubilized BFD. The fraction of HFM which was transfered to the insolubilized BFD was however almost independent from its concentration in the plasma, which indicates that the concentration of HFY cannot be determined by measuring the distribution of labeled HM. 2 to
When purified labeled anti-HFg Ig solutions were mixed with 0.25 mg insolubilized BFD containing 1 ;igHEM, 15 to 20 percent of the label remained associated with the gel after washing. This binding was specific since the labeled antibodies could be displaced by unlabeled antibodies but not by non-immune immunoglobulins and since the binding to unsubstituted BFDSepharose was low. A good correlation was found between the amounts of HEM associated with BFD-Sepharose, as determined from the binding of '*51-labeled HEM and of the binding of 1311-labeled anti-HFg Ig. When 20 ul of two normal plasmas, both with a fibrinogen level of 2.5 mg per ml, were mixed with 0.25 mg insolubilized BFD and the amount of "HFM" associated with the gel quantitated with 1311-labeled anti-HFg Ig, 0.6 and 0.8 percent of the plasma fibrinogen remained associated with the gel. Whether this amount is due to incomplete removal of fibrinogen or the presence of fibrin monomer in normal plasma or both remains unsettled. If all of this material represented soluble fibrin, its concentration in normal plasma would represent 2.5 to 3 percent. When increasing amounts of l*'I-labeled HFM were added to these normal plasmas, the amount determined with the 1311-labeled antibodies increased proportionally over the background value of the plasma. These findings indicate that HEM in human plasma can be quantitated by mixing it in a test tube with insolubilized BFD and measurements of the HFM associated with the washed gel with the use of labeled antibodies to HFg which do not cross-react with BFD. The procedure required an analysis time of about 5 hours. It should be stressed that the distribution of HFM between insolubilized BFD and soluble fibrinogen depends on the ratio of the two competing ligands. The present experimental conditions were elaborated for a ratio of 0.25 mg insolubilized BFD and 50 ng fibrinogen in solution in a reaction volume of 500 pl. The plasma‘volume used in the assay can easily be adapted to obtain a constant fibrinogen concentration : 5 pl of a sample with a fibrinogen concentration'of 10 mg/ml (very high fibrinogen level), 20 ~1 with 2.5 mg/ml (normal fibrinogen level) or 100 nl with 0.5 mg/ml (very low fibrinogen level). In this range the changes in plasma protein concentration in the reaction mixture does not appear to influence the distribution ratio of HM. As an alternative to the proposed procedure, we have also quantitated HFM in plasma by mixing it with insolubilized human fragment D followed by measurement of the adsorbed HEM with labeled antibodies to human fragment E. The preparation of labeled antibodies to human fragment E which do not crossreact with human fragment D is however more difficult than that of antibodies to human fibrinogen which do not react with BFD.
In the presen: methodological study we have performed donS?e tracer experiments with 1251-labeled HFM and 1311-labeled antibodies to HFg. Such a method is still v2ry complicated for routine use. The procedure might however be simplified to the use of 1251-labeled antibodies only. Indeed, if the molar ratio of fibrinogen to insolubilized BFD is kept at about 15, the fraction of HM which is transfered to BFD is constant (about 25 percent) and needs not to be measured separately. It is also anticipated that the preparation and stability of labeled antibodies should far exceed that of labeled HFM. Although the clinical importance of fibrin monomer quantitation remains to be further established, the developmnt of this coagulo-radioimmunometric assay could greatly facilitate the necessary clinical investigative studies. ACKNOWLEDGMENT During this study, J.F. was supported financially by the "Premio Cenit Societat Catalana de Pediatria". REFERENCES 1.
FLETCHER, A.P. and ALKJAERSIG, N. Laboratory diagnosis of intravascular coagulation. In : Recent advances in thrombosis, Poller, L. (ed.). Churchill Livingstone, Edinburgh 1973, p. 87.
2.
VON HUGO, R., STEMBERGER,A. and GRAEFF, H.
Soluble fibrin monomer complexes demonstrated by agarose gel filtration and by adsorption on insolubilized fibrinogen : a comparative study. Thromb.Diathes.haemorrh. 34, 216, 1975.
3. EDGAR, W., McKILLOP, C., HOWIE, P.W. and PRENTICE, C.R.M. Composition of soluble fibrin complexes in pre-eclampsia. Thromb.Res. IO, 567, 1977.
R. and HEENE, D.L. Affinity chromatography and 4. MATTHIAS, F.R., REINICKJZ, quantitation of soluble fibrin from plasma. Thromb.Res. IO, 365, 1977. 5. HEENE, D.L. and MATTHIAS, F.R. Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrinmonomer. Thromb.Res. 2, 137, 1973. 6. SEMERARO, N. and COLLEN, D. Affinity chromatography of soluble fibrin in human plasma on insolubilized fibrinogen and fragment D. Thromb.Res. 12, 1059, 1978. SEMERARO, N. and COLLEN, D. Affinity chromatography of soluble 7. BINI, A., fibrin in human plasma on insolubilized fibrinogen and on fragment D from fibrinogen, non cross-linked or cross-linked fibrin. Thromb.Res. 13, 443, 1978. 8. WIMAN, B. and WALLEN, P. derivative of urokinase.
Activation of human plasminogen by an insoluble Eur.J.Biochem. 36, 25, 1973.
9. COLLEN, D., KUDRYK, B., HESSEL, B. and BLOMBACK, B. Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D. J.Biol.Chem. 250, 5808. 1975. 10.
MERSKFX, C., LALEZARI, P. and .YOHNSON, A.J. A rapid, simple, sensitive =thod for measuring fibrinolytic split products in human Serum. Proc. Soc.Exp.Biol.Med. 131, 871, 1969.
CRIX.4 FOR
FIBRIZ
189
II. EDGINGTOK, T.S., PLOW, E.F., CHAVKIN, C.I., DE ?!EER, D.H. and ?“AF$!?$X%, - i R.Y. The influence of CEA-S from different tumors and of CEA as ligands on the specificity of the CEA-S radioimmunoassays. Bull.Canc. 63, 673, 1976. 12. LARGO, R., HELLER, V. and STRAUB, P.W. Detection of soluble intermediates of fibrinogen-fibrin conversion using erythrocytes coated with fibrin monomer. Blood 47, 991, 1976.
RESEAHCH 16; 1?5-189 Press Ltd.1979. Printed
in Great
Britain
A COACLZO-lUDIOIMMUNOMBTRIC ASSAY FOR THE QUANTITXTION OF FIBRIN MONOMER IN HDMAN PLASMA. PRINCIPLES AND DEVELOPZNT OF THE METHOD
3.
Felez*, E.F. Plow, B. Wiman and D. Collen
Center for Thrombosis and Vascular Research Department of Medical Research University of Leuven, Belgium
(Received
26.12.1978; in revised form 23.5.1979. by Editor M.J. Larrieu)
Accepted
ABSTRACT When a mixture of '251-labeled human fibrin monomer (HFM) and human plasma or purified fibrinogen (HFg) is added to insolubilized bovine fragment D (BFD-Sepharose), the amount of HFM which binds to BFD-Sepharose is proportional to the amount of HFM and BFD-Sepharose and inversely proportional to the amount of fibrinogen in the test tube. With 15-fold molar excess of insolubilized BFD over HFg and 2 to IO percent of HFM (compared to HFg) in the test system about 25 percent of the HFM binds to insolubilized BFD. The distribution ratio of labeled HFM is however almost independent of the concentration of HFM, and can therefore not be used to quantitate HFM in plasma. When purified 1311-labeled antibodies to HFg are added to BFDSepharose to which 1251-labeled HM is bound, the binding of 1311 is proportional to the amount of HFM on the gel. This indicates that labeled antibodies can be used to quantitate HFM bound to BFD-Sepharose. Under the experimental conditions studied (20 ~1 plasma containing 50 ug HFg and 0 to 5 ug HFM incubated with 0.25 mg insolubilized BFD), 0.6 - 0.8 percent of the plasma fibrin(ogen) and 25 percent of the HFM bind to the gel. Thus addition ?5,3_percent HFM to plasma results in a doubling of the amount of I labeled anti-HFg Ig bound to BFD-Sepharose. This test tube method for the quantitation of HFM in plasma has an analysis time of about 5 hours in its present version and a large number of samples can be handled at the same time. It may therefore constitute a useful alternative to the affinity chromatographic method for measuring HFM in plasma.
*
On leave from Servicio Hematologia, Hospital S. Pablo, Barcelona, Spain. Abbreviations used : HFg : human fibrinogen; BFg : bovine fibrinogen; HFM : human fibrin monomer; BFD : bovine fraqnen: D
CRIXA
FOR FIBRIN
VOf.l6,NO.L/’
INTRODUCTION The presence of fibrin monomer in plasma provides primary and direct evidence for the activation of the coagulation system leading to fibrin formation. Detection of fibrin, either as circulating monomer or in complexes with fibrinogen , is diagnostic of disseminated intravascular coagulation and an indicator for the “hypercoagulable state” (1). The specific detection and quantitation of fibrin monomers by affinity chromatography on insolubilized fibrinogen (2-4) is based on the original description of Heene and Matthias (5). In methodological studies (6,7) we have demonstrated that insolubilized fragment D may be substituted for insolubilized fibrinogen and constitute a more specific adsorbant for fibrin monomer from human plasma. The affinity chromatographic procedures, performed on columns, have some practical disadvantages which hamper their routine application. Large matrix to sample ratios are used to obtain near-quantitative binding of fibrin and extensive washing is required to remove co-adsorbed fibrinogen from the columns before dissociating the soluble fibrin for further analysis. Thus in general the analysis time is about one day and only a few samples can be handled at the same time. In view of these disadvantages, we have tried to elaborate a test tube method for the quantitation of soluble fibrin in human plasma. The present studies demonstrate that under appropriate conditions the distribution of fibrin monomer between soluble fibrinogen and insolubilized bovine fragment D is relatively independent of fibrin monomer concentration. This permits the use of labeled specific antibodies to human fibrinogen to precisely quantitate fibrin monomers bound to insolubilized fragment D in a highly sensitive assay system. MATERIALSAND METHODS Fibrinogen Human fibrinogen (grade L) (HFg) and bovine fibrinogen (BFg) were gifts from Kabi AB, Stockholm, Sweden (courtesy of Dr. B. Strindberg). Purification
of BFD
Two g of BFg were dissolved in 100 ml 0.05 M phosphate - 0.10 M NaCl and digested with plasmin (0.5% buffer, pH 7.5, containing 0.04% Na axide, W/W) obtained by activation of purified human plasminogen with insolubilized urokinase (8). The digestion was carried out at 4’C overnight and terminated by addition of aprotinin (Trasylol, Bayer, Leverkusen, W. Germany) (2,000 kallikrein inhibitor units (KIU) per mg plasmin). using a combination of affinity chroBFD was isolated from the digest, matography on insolubilized thrombin-treated BFg (bovine fibrin monomerSepharose) (9) followed by gel filtration on Sephadex G-150 in 0.1 M NaHC08 Sodium dodecylsulphate polyacrylamide gel electrophore- 2 M NaCl, pH 8.0. sis of the BFD preparations used in this study revealed essentially one band with a molecular weight of approximately 100,000, very similar to human fragment D previously described (9).
CRIYA
vo1.16,xo.1/2
Preparation
of
insolubilized
FOR
BFD for
FIBRIN
affinity
177
chronacography
Purified BFD was coupled to Sepharose 4 B (Pharmacia Fine Chemicals, Uppsala, Sweden) by the CNBr procedure essentially as described by Heene and Matthias (5). The substitution ratio was on average 12-14 mg BFD per ml gel (settled volume). The gel was washed with 0.05 M Tris.H3P04 - 0. I M NaC1 0.01 M EDTA - 0.001 M TAMe, buffer pH 7.4, containing 0.04% Na azide, 1X bovine serum albumin and IO KIti aprotinin/ml (buffer A), and suspended in approximately two volumes of the same buffer to obtain a BFD concentration of 5 mg per ml suspension. Purification
of
specific
antibodies
to
HFg
Rabbits were immunized by multiple site injection of 0.1 mg of purified HFg suspended in Freund adjuvant (complete adjuvant on the first injection, incomplete adjuvant on subsequent injections) at weekly intervals for three weeks. Serum obtained from late Booster injections were given bimonthly. bleedings of individual rabbits was used for isolation of the antibodies. Purified antibodies ?o HF8 which do not cross-react with BFD were isolated by absorption of the antiserum with insolubilized BFg followed by immunoabsorption on insolubilized HFg and elution of the specific antibodies, The titers of the which were finally again absorbed with insolubilized BFD. antisera were determined by hemagglutination as described by Merskey et al. using tanned red cells coated with purified HFg (I mg protein incuba(IO), ted per 100 ml cell suspension) or purified BFD (2 ag fragment D incubated with 100 ml cell suspension) . In a typical exp*riment, 50 ml of rabbit antiserum to HFg with a titer of l/8000 as assayed with red cells coated with HFg and a titer of l/500 asthrough a column (I .5 x sayed with the red cells coated with BFD, was passed 25 cd of BFg-Sepharose (substitution ratio 17.4 mg fibrinogen/ml gel), equilibrated with 0.05 M phosphate - 0.1 ?! NaCl buffer pH 7.5, containing 0.04X sodiumazide and IO KIU aprotinin/ml at room temperature and a flow rate of 40 ml per hour. The adsorbed antibodies were eluted with 0.1 M glycine. HCl pH 2.8, the column reequilibrated and the solid phase absorption repeated twice on the filtrate. after concentration to its The resulting antiserum, original volume had a titer of l/1000 assayed with red cells coated with HFr and c I/? with red cells coated with BFD. The specific antibodies to HFg were isolated from this absorbed antiserum by immunoabsorption on a column (0.9 x 12 cm) of HFg-Sepharose (substitution ratio 19 mg/ml of settled gel), using 0.1 PI glycine.HCl, pH 2.8, for dissociation of the antigen-antibody complexes. The eluted protein was dialyzed against 0.05 M phosphate - 0.1 M NaCl buffer pH 7.5, containing 0.042 sodiumazide and IO KIU aprotinin/ml, and after concentration by vacuum dialysis, applied to an Ultrogel AcA34 column (2.5 x 40 cm). The fractions containing agglutinating activity towards red cells coated with HFg were pooled and concentrated to 20 ml. This antibody solution had an absorbancy of 0.19 at 280 run, a titer of l/1000 in the assay with red cells coated i;ith HFg and a titer of < I 2 In the assay with BFD cells. The purified antibodies were labe 1 ed wi th I as described below with specific activities which attained uo to 1 mCi/m6’brotein. After iodination another absorption was performed by passing the antibody solution through a 0.9 x 3 cm column of BFD-Sepharose (Substitution ratio 13 mg per ml gel). This procedure did not affect the protein concentration or titer of the antibody solution with HFg coated red cells but reduced the binding of labeled antibodies to insolubilized BFD to less < 1 .5 percent.
Preparation
of
:iF?I
HFM was prepared by dissolving HFg in 0.25 l! phosphat2 - :?. i’? Y ::a:1 containing IO KIU aprotinin/ml, to a final con.-entra0.005 M EDTA, pH 7.5, ,_ tion of 1 to 7 mg per ml. Thrombin (Topostasine, Roche, Basel, Switzerland) per ml solution at room was added to a concentration of I to 2 NIH units temperature and the fibrin collected by winding it onto a glass rod. Xfter 30 min the clot was removed, dried on filter paper, washed twice with buffer and dissolved in 0.05 X sodium acetate - 2 X ?;aBr, buffer pH 5.3, containing 10 KIU aprotinin per nl. The dissolved fibrin xas reclotted by diluting the NaBr solution ten-fold with 0.05 ?! phosphate - 0. i !I XaCl - 0.005 ?f EDT_‘ buffer pH 7.5 and the clot was again redissolved in 0.05 X sodium acetate - 3 X SaBr buffer, pH 5.3. The fibrin concentration in the final solution was determined from its absorbancy at 280 nm (Al% = 15) and purified antithrombin III (obtained 28Q n$ from Kabi AB, Stockholm, we en) and heparin (Liquemine, Roche, Basel, Switzerland) were added in ten-fold molar excess to thrombin in order to insure inhibition of residual thrombin. 1251-labeled HFX was obtained The of labeled and unlabeled HFg. solutions averaged 20 uCi per mg.
in the specific
same way, activity
starting of the
from mixtures labeled HP>:
Radioiodination Proteins were radioiodinated by chloramine T oxidation using a reduced Initially 0.5-I .O mCi carrier free Sal251 or Xa13’I volume procedure (11). (IRE, Fleurus, Belgium) were added to 100 ug protein at 1.0 mg/ml in 0.5 11 The reaction was initiated by addition of 20 ;g sodiumphosphate pH 7.3. chloramine T and proceeded for 3 min at room temperature at which time 22 ug sodium disulfite was added followed by RI and bovine serum albumin to final ‘*‘I or 1311 were removed by exconcentrations of 0.1 mg per ml. Unbound Specific activities of 1 mCi/mg were routinally achieved tensive dialysis. and precipitability of radioactivity in 10% trichloroacetic acid generally exceeded 97%. Quantitation solubilized
of the BFD
distribution
of
labeled
HFX between
soluble
HFg and
in-
the distribution of HFM between soluble HFg and Unless stated otherwise, insolubilized BFD was measured by adding successively to 6 ml polystyrene tubes : 1) 10 to 200 ul titrated human plasma or purified HFg solution 2) 20 ~1 of a mixture of labeled and (fibrinogen concentration 2.5 mg/ml), unlabeled HFX dissolved in 0.05 M acetate - 2 Y NaBr pH 5.3 (amount of fibrin incubation for I hour at room temperature sufadded 0.5 to IO ug) , 3) after ficient diluent consisting of 0.05 M Tris.H PO4 - 0.10 M NaCl - 0.01 ?! EDTA IX bovine serun albumin - 0.001 M TAMe, pH 7.4, containing 0.04% so a-iumazide, and IO KIU aprotinin per ml (buffer A) was added to obtain a final volume of 0.5 ml, and 4) after 10 min incubation 50 to 200 ul suspension of BFD-Sephacontaining 5 mg of BFD per ml suspension. rose, The mixture was stirred for I hr at room temperature and then centriThe gel was washed 5 times by suspending it in fuged for 2.5 min at 3000 g. 1 ml buffer A and stirring for approximately 5 min at room temperature. The HFM which remained ass?Siated with the insolubilized BFD was then calculated During the 4th and the 5th wash, IO to 20 percent from measurements of I. of the radioactivity which remained associated with the gel was removed.
CRI%
Quantitation of EM, to HFg.
FOR
FIBRIN
bound to insolubilized 8FD ;lith '3'I-labeled antibodies
The HFH containing BFD-Sepharose was resuspended in 0.: ml buffer A, 100 21 of a mixture of !3iI-labeled and unlabeled antibodies to HFg was added and the mixture stirred for I hr at room temperature. The labeled antibodies were diluted with unlabeled antibodies in such a vay that about I5 percent of the label (1500 cpm) was bound by 0.25 mg insolubilized BFD containing 1 ug HFX. The mixture was stirred for I hour at room ;;yperature and I. Control the gel ijaswashed 5 times with I ml buffer to remove unbound experiments without RFg, HFX or both added to the BFD-Sepharose were run simultaneously. With the fifth wash only negligible amounts of 13'1 were eliminated. Labeled antibody displacement curves The optimal dilution of labeled with unlabeled antibodies for the quantitation of HFM bound to insolubilized BFD was determined in the following way. Standard amounts of HFN bound to BFD-Sepharose (0.25 mg BFD) were incubated with 1311-labeled antibodies to HFg which were progressively diluted in cold antibodies. The HFN substituted BFD-Sepharose was obtained by incubating l251-labeled HFM in the absence of HFg with BFD-Sepharose and the amount of HFM bound to BFD calculated from 1251 measurements. Binding ratios of 1.0, 0.5 and 0.25 pg of HFX per 0.25 mg of insolubilized BFD were analyzed. The gel was suspended in sufficien t bclffer A to obtain a final volume .>f0.50 ml, the mixture of labeled (representing 10,000 cpm) and unlabeled antibodies was added and the suspension stirred for one hr at room temperature. The gel was then isolated by centrifugation, washed 5 times with I ml volumes of buffer A and the '311 associated with the gel was then measured. Recycling of BFD-Sepharose 125 I-labeled HFM and BFD-Sepharose which had been in contact with both i311-labeled anti-HFg Ig was collected and stored in buffer A. When sufficient gel had accumulated, it was poured in a column and washed with 0.05 M sodium acetate - 2 m NaBr buffer pH 5.3 for at least 24 hr. This resulted in removal of 80-90 percent of both 1251 and I311 associated with gel without loss of binding capacity for 125I-HFX. RESULTS Distribution of human fibrin monomer between soluble HFg and insolubilized BFD When 20 ~1 mixtures of labeled and unlabeled (0.5 to 2.5 ug) HFM were added to 200 ~1 BFD-Sepharose (1 mg BFD) suspension in a total volume of 0.5 ml and the suspension stirred for I hr at room temperature about 90 per-cent of the labeled material remained associated with the gel after washing (Fig. IA). When 200 pl of purified HFg solution or human plasma were preincubated for I hr at room temperature with 20 ul mixtures of labeled and unlabeled HFM (1 to 10 ug) and then the BFD-Sepharose was added, the percentage of labeled material which bound to BFD decreased with increasing amounts of HFM and moreso with increasing concentration of HFg (Fig. IA). The total amount of HFM which remained associated with the insolubilized BFD, calculated from the experiments in Fig. IX is represented in Fig. iB. The results demonstrate that the amount of HM associated with the BFD was
CRI;ttl.
189
proportional concentration. solubilized
FOR
TiOl.lS,SO.l/2
FIBRI?;
to
the HE?! concentration and inversely The latter findings indicate that BFD compete with each other for !!FX.
?ro?or:i~al the soluble
Wg
to the :iCg and the in-
60 I
OL
G
1
3 HFM
3
lo ADDED
HFM
(~91
FIG.
la ADDED
(w)
I
Binding of fibrin monomer to insolubilized bovine fragment D. Experimental conditions : 200 ul fibrinogen solution of different concentra tion was incubated with 20 ul of a mixture of labeled and unlabeled fibrin 1 hr at room temperature. Then 200 ul suspension of insolubimonomer for lized fragment D (representing 1 mg fragment D) was added and after incubation for I hr the unbound label was removed by washing the gel 5 times with 1 ml buffer. The amount of radioactivity associated with the gel was measured and expressed as percentage of the total (A) and converted into ug human fibrin monomer bound (B) . The data represent means + SD from four separate experiments. The amount of HFM associated with the gel exhibited a linear dependence on the total amount of insolubilized BFD present (Fig. 2). This relationship was only marginally affected by fibrinogen concentration. The influence of the incubation time on the binding of fibrin monomer is shown in Fig. 3. Maximum binding was obtained after about 2 hours and remained constant between 2 and 4 hours.
CRI?Q~ FOR
I
0.25 INSOLUBILIZED
1Sl
FIBRIX
I
I
0.50 8FD
1 (mg)
FIG. 2 Influence of the amount of insolubilized bovine fragment D on the binding of human fibrin monomer. Experimental conditions : IO0 ~1 fibrinogen solution was incubated with IO ul mixtures of labeled and unlabeled fibrin monomer for 1 hr at room temperature. Then buffer and insolubilited bovine fragment D suspension were added to a final volume of 0.5 ml. The total amount of gel in each tube was adjusted with non-substituted gel to a volume of 200 ~1. (*I : 3 ug fibrin monomer and 500 pg fibrinogen in the assay system (a) : 3 up fibrin monomer and 250 ug fibrinogen (0) : IO ug fibrin monomer and 500 u'gfibrinogen in the assay system @I) : 10 g fibrin monomer and 250 ug fibrinogen
These results establish the characteristics of the affinity system when an initial distribution between '251-HFM and HFM and fibrinogen is permitted prior to addition of the BFD-Sepharose. Under these conditions, binding of HFM to the beads is dependent upon : a) HFM, b) fibrinogen, c) BFD beads, and d) time. In plasma samples for analysis, this condition would, however, not exist since an initial interaction of HFM in the plasma with plasma fibrinogen would occur prior to the addition of the '251-HFM. Therefore, the characteristics of the affinity system was examined whe HFX and fibrinogen were preincubated for one hour prior to the addition of iY5I-HFM. The results shown in Figure 4 demonstrate that such preincubation markedly affects the data observed. With such preincubation, the binding of labeled HFM to the EFD beads was no longer dependent upon HFM or fibrinogen concentration. This is in marked contrast to the results observed in Figure IA where, without preincubation, binding was dependent upon both these variables. Presumably, the dissociation constant of H Py5ard fibrinogen is low, and an effective reequilibration between HFM and I HFM is not achieved within one hour. Prolonged incubation with '251-HFM or high concentrations of the label should yield results similar to Figure IA but that would preclude a rapid or sensitive analysis. Therefore, it does not appear possible to quantitate HF?! in plasma by the addition of trace amounts of labeled HFY.
CRIXk
182
FOR
FIBRIX
VOl.lb,XO.l/2
/
g
;/* I / olY~ 1
??
INCUBATION FIG.
4
2 TIME
IN
HOURS
3
Influence of incubation time on the binding of labeled fibrin monomer to insolubilized bovine fragment D. Experimental conditions : 100 ~1 fibrinogen solution was premixed with IO ~1 labeled fibrin monomer for I hr at room bovine fragment D and buffer up to a temperature. Then I mg insolubilized final volume of 0.5 ml was added. After different incubation times the gel was washed 5 times and the amount of fibrin monomer associated with the gel quantitated by isotope measurements. monomer and 1500 pg fibrinogen in the assay (*I : IO ng fibrin 150 ug fibrinogen in the assay I ug fibrin monomer and (0) :
Quantitation of HFM, adsorbed specific antibodies to HFg
to
insolubilized
Addition of labeled HFM cannot Therefore a radioimmunometric assay adsorbed HFM.
BFD with
be used to measure was elaborated for
the
use
of
labeled
HFM bound to BFD. the quantitation of
Antibodies raised against HFg were immunochemically purified on insolunon-reactive with insolubilized BFD as described bilized HFg, and rendered When these labeled antibodies were added to under naterials and methods. 0.25 mg insolubilized HFg, between 30 and 60 percent was bound, whereas under comparable experimental conditions only I to 1.5 percent bound to 0.25 mg insolubilized BFD and 0.4 to 0.7 percent associated with an equivalent The antibodies therefore seem to be specific amount of unsubstituted gel. enough for quantitation of the HFM-bound to insolubilized BFD.
.
For quantitative analysis, labeled antibody displacement curves were constructed using HFM bound to insolubilized BFD as ligand and 1311-labeled anti-HFg Ig diluted with unlabeled antibodies as described under materials The results obtained in a typical experiment are shown in and methods Whereas 60 percent of the labeled antibodies bound to 250 ug HFM, Fig. 5. about 22 percent was associated with the BFD gel containing I u!p,9FM and 11
CRIMA
vo1.16,80.1/‘2
FOR
133
FIBRIS
1000 pg Fg f 270 rrg Fg f
, 1
I 3
10 HFM
ADDED
FIG.
(pg)
4
Binding of fibrin monomer to insolubilized bovine fragment D. Sxpcrlmental conditions : 200 ul fibrinogen solution of different concentration was incubated with IO ul of unlabeled fibrin monomer for 1 hr followed by addition of a trace amount of labeled fibrin monomer and further incubation I hr. for Then 200 ~1 suspension of insolubilited fragment D (representing 1 mg fragment D) was added and after incubation for I hr the unbound label was The amount of radioacremoved by washing the gel 5 times with I ml buffer. tivity associated with the gel was measured and expressed as percentage of the total. The data represent means -+ SD from four separate experiments.
percent with the gel containing 0.25 ug HF!+. These values are corrected for the binding of I .5 percent 1311 to 0.25 mg insolubilized BFD. Non-immune Ig did not cause displacement of labeled antibodies indicating that their binding is due to reaction with adsorbed HFM. For subsequent quantitation of HFX in plasma or purified fibrinogen solutions a labeled antibody dilution of 3.5 times was used. Calibration curves for the quantitation of HM bound to BFD-Sepharose were constructed by incubation of increasing amounts of a mixture of ‘251labeled and unlabeled liF!I (0.25 to 7 -g.) with 0.25 mg insolubilized BFD.
In
CRIWL
b
I
1 1.5 DILUTION
FOR
FIBRIS
t
3.5 OF 13’1 LABELED
13.5 ANTI- HFG
lg
FIG. 5 Antibody displacement curves of 1311-labeled anti-HFg Ig from HFM bound to BED-Sepharose. Experimental conditions : 0.25 mg insolubilized BFD to which 0.25 to 1 ng ~~~n:~In~~~n~ow~~oi~~~bt5~d with dilutions of.10 ~1 1311-labeled anti-HFg Ig I) in unlabeled antibodies for I hr at room temperature in a f&al volume of 0.5 ml. The unbound label was removed by 5 washings with 1 ml buffer and the '3*1 quantitated.
the absence of fibrinogen in the solution 70 to 85 percent of this material bound to the BFD-Sepharose. The washed gels were then incubated with labeled antibodies and the percent 13'1 bound quantitated as described above. An almost linear relationship was found between the log of HFM bound to BFDSepharose and the percentage labeled antibodies bound (Fig. 6). HFM was quantitated in human plasma in the following way. Fresh human plasma from two healthy subjects with a fibrinogen concentration of 2.5 mg per ml (20 ul) was mixed with 10 ul 0.05 M sodium acetate - 2 M NaBr pH 5.3 containing a mixture of labeled and unlabeled HFM (0 to 5 ug HFM) and 0.42 ml buffer A was added followed after approximately 10 min incubation by 50 ~1 BFD-Sepharose suspension (0.25 mg BFD). The mixture was stirred for I hr at room temperature and the gel washed 5 times for 5 min at room temperature with I ml buffer A. Incubation in the presence of 0.4 M urea or 0.i.M NaBr, or washing with buffer A with pH adjusted to 6.0 or 8.0 ot with buffer A containing I M NaCl did not alter the results significantly.
CRI?l.A
Vol.lf,So.1/2
FOR
FIBRIS
1Sj
T
I-
c-
I
I
.
0.1
02 HFM
a5 BOUND
1 TO
2
5
BFD-SEPHAROSE
10 tyg)
131 Quantitation of “FM bound to BFD-Sepharose with I-labeled anti-HFg Ig. Experimental conditions : see text. o and * represent curves obtained with antibodies purified from two different antisera; the data represent means + SD from triplicate runs.
The washed gels were suspended in 0.5 ml buffer A and a mixture of ‘3’1_ labeled (10,000 cpm) and unlabeled (dilution factor 3.5) anti-HFg Ig addfg’ The suspension was stirred for I hr at room temperature and the unbound I removed by washing the gel 5 times with I ml buffer for 5 min at room temperature. The bound isotope was then measured and the corresponding amount of HFM bound to BFD-Sepharose calculated from the calibration curve in Fig. 6. The results obtained with the plasma from two healthy syjjects are summarized in Fig. 7. In the absence of added HFM the amount of I bound to the gel corresponded to 0.3 and 0.4 ug, representing 0.6 to 0.8 percent of the fibrinogen. In the presence of added ‘251-labeled HFM the W3 bound to the gel increased proportionally to the amount of I-HFM associated with the gel and corresponded to the amount calculated from the calibration
i-01.
p
;6 ,x0.1/2
5d
!/ 8 z s Y G
0
0.5
ID
12’1 HFM-BOUND
15
TO BFD-SEPHAROSE
(m)
FIG.? Correlation between the amount of soluble fibrin in human plasma determined from measurements of added 1251-labeled HFM and from the binding of 13'1labeled anti-HFg Ig. The data are obtained with plasma from two different healthy subjects.
curve in Fig. 6 (the lines in Fig. 7 are drawn at an angle of 45"). These findings suggest that the quantitation of soluble fibrin in human plasma by way of I-labeled antibodies to HFg following adsorption of the plasma with insolubilized BFD may be a valid approach. DISCUSSION Affinity chromatography on insolubilized fibrinogen has been used for the quantitation of soluble fibrin in human plasma. This analysis may be of value for the diagnosis of in vivo coagulation but its clinical application is limited by the fact that column chromatography is time consuming and only one sample can be analyzed per column at one time. In order to circumvent these disadvantages we have tried to elaborate a test tube version based on the affinity between fibrin monomer and high molecular weight fragment D (6). As one might expect, the soluble fibrinogen in plasma competed with the insolubilized fragment D for the fibrin monomer resulting in incomplete transfer of fibrin monomer from fibrinogen to the insolubilized fragment D. Such competition between insolubilized fibrin monomer and soluble fibrinogen for fibrin monomer present in plasma has previously been described by Largo et al. (12). At a 15-fold molar excess of fragment D over fibrinogen which contained
Vol.f6,?io.1/2
CRI?U. FOR
FIBRIN
18;
1251-labeled fibrin monomer, approximately 25 percent of the 10 percent fibrin monomer was transfered to the insolubilized ligand whereas less than 1 percent of the fibrinogen remained associated with the gel after washing. No differences were observed between insolubilized human fragment D (data not reported) or bovine fragment D (BFD). The transfer of human fibrin monomer (HEM) to BFD thus is sufficiently high and the binding of human fibrinogen (HFg) sufficiently low to allow quantitation of HFX in plasma from the amount bound to insolubilized BFD. The fraction of HFM which was transfered to the insolubilized BFD was however almost independent from its concentration in the plasma, which indicates that the concentration of HFY cannot be determined by measuring the distribution of labeled HM. 2 to
When purified labeled anti-HFg Ig solutions were mixed with 0.25 mg insolubilized BFD containing 1 ;igHEM, 15 to 20 percent of the label remained associated with the gel after washing. This binding was specific since the labeled antibodies could be displaced by unlabeled antibodies but not by non-immune immunoglobulins and since the binding to unsubstituted BFDSepharose was low. A good correlation was found between the amounts of HEM associated with BFD-Sepharose, as determined from the binding of '*51-labeled HEM and of the binding of 1311-labeled anti-HFg Ig. When 20 ul of two normal plasmas, both with a fibrinogen level of 2.5 mg per ml, were mixed with 0.25 mg insolubilized BFD and the amount of "HFM" associated with the gel quantitated with 1311-labeled anti-HFg Ig, 0.6 and 0.8 percent of the plasma fibrinogen remained associated with the gel. Whether this amount is due to incomplete removal of fibrinogen or the presence of fibrin monomer in normal plasma or both remains unsettled. If all of this material represented soluble fibrin, its concentration in normal plasma would represent 2.5 to 3 percent. When increasing amounts of l*'I-labeled HFM were added to these normal plasmas, the amount determined with the 1311-labeled antibodies increased proportionally over the background value of the plasma. These findings indicate that HEM in human plasma can be quantitated by mixing it in a test tube with insolubilized BFD and measurements of the HFM associated with the washed gel with the use of labeled antibodies to HFg which do not cross-react with BFD. The procedure required an analysis time of about 5 hours. It should be stressed that the distribution of HFM between insolubilized BFD and soluble fibrinogen depends on the ratio of the two competing ligands. The present experimental conditions were elaborated for a ratio of 0.25 mg insolubilized BFD and 50 ng fibrinogen in solution in a reaction volume of 500 pl. The plasma‘volume used in the assay can easily be adapted to obtain a constant fibrinogen concentration : 5 pl of a sample with a fibrinogen concentration'of 10 mg/ml (very high fibrinogen level), 20 ~1 with 2.5 mg/ml (normal fibrinogen level) or 100 nl with 0.5 mg/ml (very low fibrinogen level). In this range the changes in plasma protein concentration in the reaction mixture does not appear to influence the distribution ratio of HM. As an alternative to the proposed procedure, we have also quantitated HFM in plasma by mixing it with insolubilized human fragment D followed by measurement of the adsorbed HEM with labeled antibodies to human fragment E. The preparation of labeled antibodies to human fragment E which do not crossreact with human fragment D is however more difficult than that of antibodies to human fibrinogen which do not react with BFD.
In the presen: methodological study we have performed donS?e tracer experiments with 1251-labeled HFM and 1311-labeled antibodies to HFg. Such a method is still v2ry complicated for routine use. The procedure might however be simplified to the use of 1251-labeled antibodies only. Indeed, if the molar ratio of fibrinogen to insolubilized BFD is kept at about 15, the fraction of HM which is transfered to BFD is constant (about 25 percent) and needs not to be measured separately. It is also anticipated that the preparation and stability of labeled antibodies should far exceed that of labeled HFM. Although the clinical importance of fibrin monomer quantitation remains to be further established, the developmnt of this coagulo-radioimmunometric assay could greatly facilitate the necessary clinical investigative studies. ACKNOWLEDGMENT During this study, J.F. was supported financially by the "Premio Cenit Societat Catalana de Pediatria". REFERENCES 1.
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